Degeneres voices the lead character of Dory, a fish with short-term memory loss who appeared in the movie's 2003 prequel "Finding Nemo."
Rumours have swirled about the film after the release of the trailer, which apparently showed a lesbian couple pushing a stroller.
And DeGeneres, 58, joked that she was unaware of any homosexual themes within the movie, reported USA Today.
"I don't know if it's true, I don't know if there is, or if she just has a bad short haircut. Who knows if she's a lesbian?," she told USA Today of the rumoured gay character.
The highly-anticipated Pixar film follows Dory's journey as she tries to find her family, and features the vocal talents of Eugene Levy, Diane Keaton, Dominic West, Ty Burrell and Idris Elba.
DeGeneres, who returns to the sequel opposite "Finding Nemo" co-star Albert Brooks, says "Finding Dory" is much darker than the 2003 prequel.
"I think it is both darker and lighter. It's more complex and layered, and there's more messaging in it. And it's still obviously a kid's film, and it's an animated Disney Pixar film, but it's more... I think adults will be equally, if not more, drawn to this film," she said.
And, when asked if she was anything like her character Dory, the comedienne replied, "I think I, like Dory, feel different. I think I probably don't think the way everybody else thinks, and so there's that.
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